Therapeutic application of extracellular vesicle-encapsulated CC16 in acute lung injury
Authors: Lanzhi Liu, Juan Fandiño, Daniel P. O’Toole
Journal: Molecular Therapy (2023)

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a critical clinical syndrome spectrum with high incidence and associated mortality. It is characterized by acute hypoxemia, reduced lung compliance, and non-cardiogenic pulmonary edema, which lead to respiratory failure.1Butt Y. Kurdowska A. Allen T.C. Acute lung injury: a clinical and molecular review.Arch. Pathol. Lab. Med. 2016; 140: 345-350Crossref PubMed Scopus (552) Google Scholar,2Matthay M.A. Zemans R.L. Zimmerman G.A. Arabi Y.M. Beitler J.R. Mercat A. Herridge M. Randolph A.G. Calfee C.S. Acute respiratory distress syndrome.Nat. Rev. Dis. Primers. 2019; 5: 18Crossref PubMed Scopus (646) Google Scholar Despite considerable progress in supportive treatment for ALI/ARDS, effective pharmacological interventions have not been developed. In recent years, therapies using extracellular vesicles (EVs) are becoming more popular, and there is strong interest in modifying EVs to enhance their targeting efficiency and their content with beneficial cargos. To date, evidence from several preclinical and clinical studies has shown that EVs play important roles both in inducing and attenuating ALI/ARDS.3Mahida R.Y. Matsumoto S. Matthay M.A. Extracellular vesicles: a new frontier for research in acute respiratory distress syndrome.Am. J. Respir. Cel Mol. Biol. 2020; 63: 15-24Crossref PubMed Scopus (42) Google Scholar In this issue of Molecular Therapy, Han et al.4Han Y. Zhu Y. Almuntashiri S. Wang X. Somanath P.R. Owen C.A. Zhang D. Extracellular vesicle-encapsulated CC16 as novel nanotherapeutics for treatment of acute lung injury.Mol. Ther. 2023; (Preprint at)https://doi.org/10.1016/j.ymthe.2023.01.009Abstract Full Text Full Text PDF Scopus (3) Google Scholar report a new approach for the treatment of ALI by using small extracellular vesicles (sEVs) loaded with the anti-inflammatory protein club cell protein 16 (CC16). sEV-CC16 attenuated lipopolysaccharide (LPS) and bacteria-induced ALI in wild-type and CC16 knockdown mice through modulating inflammatory response and DNA repair pathways. Compared with recombinant CC16 (rCC16), sEV-CC16 presented more therapeutic potential even at lower doses. These findings have deepened our understanding of the potential benefit in ALI/ARDS and have provided an innovative new therapeutic choice for ALI/ARDS by combining EVs and anti-inflammatory factors. CC16 secreted by pulmonary club cells has been proposed as a biomarker of lung epithelial injury, and the level of CC16 is reduced in plasma and pulmonary edema fluid from ALI patients.5Kropski J.A. Fremont R.D. Calfee C.S. Ware L.B. Clara cell protein (CC16), a marker of lung epithelial injury, is decreased in plasma and pulmonary edema fluid from patients with acute lung injury.Chest. 2009; 135: 1440-1447Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar However, the association between CC16 and EVs has remained unclear, and the level of EV-derived CC16 has not yet been identified. Han et al.4Han Y. Zhu Y. Almuntashiri S. Wang X. Somanath P.R. Owen C.A. Zhang D. Extracellular vesicle-encapsulated CC16 as novel nanotherapeutics for treatment of acute lung injury.Mol. Ther. 2023; (Preprint at)https://doi.org/10.1016/j.ymthe.2023.01.009Abstract Full Text Full Text PDF Scopus (3) Google Scholar demonstrated that CC16 protein is carried mainly in small EVs rather than in medium or large EVs, and sEV-derived CC16 is decreased in pneumonia patients and in LPS-induced animal models of ALI/ARDS. Therefore, the authors focused on sEV-derived CC16 and generated CC16-loaded sEVs for animal studies by using the immortalized lung epithelial cell line BEAS-2B, which was genetically modified to express the protein CC16. Several different strategies can be used for engineering modified EVs, such as genetic manipulation of the parental cells, exogenous stimulation, and external stimulation.6Man K. Brunet M.Y. Jones M.C. Cox S.C. Engineered extracellular vesicles: tailored-made nanomaterials for medical applications.Nanomaterials (Basel, Switzerland). 2020; 10: 1838Crossref PubMed Scopus (59) Google Scholar The use of engineered EVs for the treatment of ALI/ARDS has not been explored in depth. Currently, only one preclinical study has used engineered EVs for the treatment of this pathology. The study demonstrated that the genetic overexpression of the protein syndecan-1 in endothelial cells increased the encapsulation into their derived EVs; moreover, the administration of these EVs in a murine model of LPS-induced ALI ameliorated lung inflammation and edema.7Zhang C. Guo F. Chang M. Zhou Z. Yi L. Gao C. Huang X. Huan J. Exosome-delivered syndecan-1 rescues acute lung injury via a FAK/p190RhoGAP/RhoA/ROCK/NF-κB signaling axis and glycocalyx enhancement.Exp. Cel Res. 2019; 384111596Crossref Scopus (39) Google Scholar To evaluate the therapeutic potential of sEV-CC16, Han et al.4Han Y. Zhu Y. Almuntashiri S. Wang X. Somanath P.R. Owen C.A. Zhang D. Extracellular vesicle-encapsulated CC16 as novel nanotherapeutics for treatment of acute lung injury.Mol. Ther. 2023; (Preprint at)https://doi.org/10.1016/j.ymthe.2023.01.009Abstract Full Text Full Text PDF Scopus (3) Google Scholar compared the effects of vehicle, sEV-control, sEV-CC16, and rCC16 in both LPS and Klebsiella pneumoniae-induced mouse models of ALI/ARDS. Through intratracheal instillation, sEV-CC16 labeled with PKH26 were effectively delivered into lung and then captured by lung macrophages and epithelial cells, mediating anti-inflammatory effects. Compared with vehicle and sEV-control, sEV-CC16 as well as rCC16 attenuated lung inflammation and lung injury, reducing the bronchoalveolar lavage fluid (BALF) count of macrophages and neutrophils, decreasing the level of proinflammatory factors (IL-1β, TNF-α, CXCL1, and CXCL2), and ameliorating lung exudate. Moreover, the transcription profile results from LPS-treated THP-1 macrophages show that sEV-CC16 not only inhibited the expression of proinflammatory genes but also activated DNA repair-related pathways. With additional in vitro studies, they discovered that sEV-CC16 suppressed LPS-induced DNA damage in both macrophages and epithelial cells, but rCC16 worked in macrophages only. The results from CC16 knockout (KO) mice in vivo were consistent with the in vitro studies. Furthermore, they investigated the underlying mechanism whereby CC16 suppresses DNA damage. They found that CC16 can block the interaction between heat shock protein 60 (HSP60) and inhibitor of κB kinase α (IKKα), which ameliorated DNA damage by inhibiting the HSP60-IKKα-NF-κB pathway. Although this pathway has not been explored in macrophages or epithelial cells from an animal model, the evidence is quite strong to support further studies. These findings support sEVs as a powerful tool to effectively deliver CC16 into target macrophages and epithelial cells in ALI/ARDS models. The characterized sEV-CC16 protect against lung injury by participating in anti-inflammatory effects and DNA repair. As sEV-CC16 are internalized via fusion with the cell membrane independent of cell receptors, they are more efficient than rCC16. The successful application of sEV-CC16 in ALI/ARDS models offers new insights into the therapeutic potential of EVs. EVs from different sources can contain pro- or anti-inflammatory factors, regulating different kinds of alveolar cells, including epithelial cells, endothelial cells, macrophages, and neutrophils.8Hu Q. Zhang S. Yang Y. Yao J.Q. Tang W.F. Lyon C.J. Hu T.Y. Wan M.H. Extracellular vesicles in the pathogenesis and treatment of acute lung injury.Mil. Med. Res. 2022; 9: 61PubMed Google Scholar Although the characterization of sEV-CC16 is well described, the immortalized cell source is not recommended for clinical therapy. Moreover, the sEV-control did not exhibit therapeutic effects, suggesting that sEVs themselves did not act as a functional factor. EVs derived from mesenchymal stromal cells (MSCs) have potential in the treatment of ALI/ARDS, attenuating lung injury through the effects of anti-inflammation, antimicrobial, and cell injury repair.9Sun H. Zhang T. Gao J. Extracellular vesicles derived from mesenchymal stem cells: a potential biodrug for acute respiratory distress syndrome treatment.BioDrugs. 2022; 36: 701-715Crossref PubMed Scopus (4) Google Scholar For further research, MSC-derived EVs could be a superior carrier for CC16. In conclusion, the findings of Han et al.4Han Y. Zhu Y. Almuntashiri S. Wang X. Somanath P.R. Owen C.A. Zhang D. Extracellular vesicle-encapsulated CC16 as novel nanotherapeutics for treatment of acute lung injury.Mol. Ther. 2023; (Preprint at)https://doi.org/10.1016/j.ymthe.2023.01.009Abstract Full Text Full Text PDF Scopus (3) Google Scholar provide significant evidence for sEV-CC16 as a potential therapy for ALI/ARDS and indicate the underlying mechanism of CC16 modulating DNA repair pathways. These findings broaden the significance of EVs as an attractive candidate for ALI/ARDS treatment and provide more possibilities for the therapeutic potential of CC16. However, important gaps remain before clinical use can be considered, such as EV safety concerns, CC16 dosing, and immune responses. Thus, further research is needed to find an optimized application of sEV-CC16 in subsequent clinical trials. The authors declare no competing interests.